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1.  Association between MTHFD1 G1958A Polymorphism and Neural Tube Defects Susceptibility: A Meta-Analysis 
PLoS ONE  2014;9(6):e101169.
The methylenetetrahydrofolate dehydrogenase (MTHFD1) gene, as one of the key genes involved in the folate pathway, has been reported to play a critical role in the pathogenesis of neural tube defects (NTDs). However, the results of published studies are contradictory and inconclusive. Thus, this meta-analysis aimed to evaluate the effect of the common polymorphism in the MTHFD1 gene, the G1958A (R653Q, dbSNP ID: rs2236225) variant, on the risk of NTDs in all eligible studies.
Relevant literature published before January 3, 2014 was retrieved from the MEDLINE, EMBASE, Cochrane Library, and CBM databases. Pooled crude odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were calculated to evaluate the association between the MTHFD1 G1958A polymorphism and NTDs risk.
We performed a meta-analysis of nine studies with a total of 4,302 NTDs patients and 4,238 healthy controls. Our results demonstrated a significant correlation between the MTHFD1 G1958A polymorphism and NTDs in an overall meta-analysis. For family-based studies, the study subjects were classified as NTD cases, mothers with NTDs offspring, and fathers with NTDs offspring. We found no association between any of the fathers’ genotypes and NTDs, whereas there was a clear excess of the 1958A allele in the mothers of children with NTDs compared with controls individuals.
In summary, our meta-analysis strongly suggests that the MTHFD1 G1958A polymorphism might be associated with maternal risk for NTDs in Caucasian populations. However, the evidence of this association should be interpreted with caution due to the selective nature of publication of genetic association studies.
PMCID: PMC4076264  PMID: 24977710
2.  MTHFR rs1801133 C>T polymorphism is associated with an increased risk of tetralogy of Fallot 
Biomedical Reports  2014;2(2):172-176.
Abnormal folate metabolism and common variants of folate-metabolizing enzymes have been described as possible risk factors for congenital heart disease (CHD). Two important folate-metabolizing enzymes involved in the folate/homocysteine metabolic pathway are 5,10-methylenetetrahydrofolate reductase (MTHFR) and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1). MTHFR and MTHFD1 polymorphisms may be associated with CHD susceptibility. To evaluate the impact of MTHFR and MTHFD1 single-nucleotide polymorphisms (SNPs) on CHD susceptibility, we genotyped functional MTHFR SNPs rs1801133 C>T, rs1801131 A>C and rs2274976 G>A, and MTHFD SNPs rs2236225 C>T, rs1950902 G>A and rs1076991 A>G in a hospital-based case-control study of 173 tetralogy of Fallot (TOF) cases and 207 non-CHD controls. When MTHFR rs1801133 CC homozygote genotype was used as the reference group, the TT genotype was associated with a significantly increased risk for TOF [TT vs. CC: odds ratio (OR)=1.67; 95% confidence interval (CI): 1.01–2.75; P=0.046]. In the recessive model, when MTHFR rs1801133 CC/CT genotype was used as the reference group, the TT homozygote genotype was associated with a significantly increased risk for TOF (OR=1.81, 95% CI: 1.15–2.84; P=0.010). In conclusion, our findings suggest that MTHFR rs1801133 C>T polymorphism may play a role in susceptibility for TOF. Large-scale studies with a more rigorous study design including diverse ethnic populations are required to confirm these findings.
PMCID: PMC3917760  PMID: 24649091
5,10-methylenetetrahydrofolate reductase; congenital heart disease; polymorphisms; tetralogy of Fallot; molecular epidemiology
3.  A Common Variant in MTHFD1L is Associated with Neural Tube Defects and mRNA Splicing Efficiency 
Human mutation  2009;30(12):1650-1656.
Polymorphisms in folate-related genes have emerged as important risk factors in a range of diseases including neural tube defects (NTDs), cancer and coronary artery disease (CAD). Having previously identified a polymorphism within the cytoplasmic folate enzyme, MTHFD1, as a maternal risk factor for NTDs; we considered the more recently identified mitochondrial paralogue, MTHFD1L as a candidate gene for NTD association. We identified a common deletion/insertion polymorphism, rs3832406, c.781-6823ATT(7-9), that influences splicing efficiency and is strongly associated with NTD risk. Three alleles of rs3832406 were detected in the Irish population with varying number of ATT repeats; Allele 1 consists of ATT7, while Alleles 2 and 3 consist of ATT8 and ATT9 respectively. Allele 2 of this triallelic polymorphism showed a decreased case risk as demonstrated by case-control logistic regression (P= 0.002) and by transmission disequilibrium test (TDT) (P= 0.001); while Allele 1 showed an increased case risk. Allele 3 showed no influence on NTD risk and represents the lowest frequency allele (0.15). Additional SNP genotyping in the same genomic region provides additional supportive evidence of an association. We demonstrate that two of the three alleles of rs3832406 are functionally different and influence the splicing efficiency of the alternate MTHFD1L mRNA transcripts.
PMCID: PMC2787683  PMID: 19777576
MTHFD1L; NTD; Splicing; Polymorphism; Association; Folate; Mitochondria
4.  Prognostic significance of folate metabolism polymorphisms for lung cancer 
British Journal of Cancer  2007;97(2):247-252.
Functional nonsynonymous single-nucleotide polymorphisms (nsSNPs) of folate metabolism genes can influence the methylation of tumour suppressor genes, thereby potentially impacting on tumour behaviour. To investigate whether such polymorphisms influence lung cancer survival, we genotyped 14 nsSNPs mapping to methylene-tetrahydrofolate reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR); DNA methyltransferase (DNMT2), methylenetetrahydrofolate dehydrogenase (MTHFD1) and methenyltetrahydrofolate synthetase (MTHFS) in 619 Caucasian women with incident disease, 465 with non-small cell (NSCLC) and 154 with small cell lung cancer (SCLC). The most significant association detected was with MTHFS Thr202Ala, with carriers of variant alleles having a worse prognosis (hazard ratio (HR)=1.49; 95% confidence interval: 1.14–1.94). Associations were also detected between overall survival (OS) in SCLC and homozygosity for MTHFR 222Val (HR=1.92; 1.03–3.58) and between OS from NSCLC and MTRR 175Leu carrier status (HR=1.36; 1.06–1.75). While there is evidence that variation in the folate metabolism genes may influence prognosis from lung cancer, current data are insufficiently robust to distinguish individual patient outcome.
PMCID: PMC2360297  PMID: 17533396
lung cancer; SNP; prognosis; folate metabolism
5.  Evaluation of common genetic variants in 82 candidate genes as risk factors for neural tube defects 
BMC Medical Genetics  2012;13:62.
Neural tube defects (NTDs) are common birth defects (~1 in 1000 pregnancies in the US and Europe) that have complex origins, including environmental and genetic factors. A low level of maternal folate is one well-established risk factor, with maternal periconceptional folic acid supplementation reducing the occurrence of NTD pregnancies by 50-70%. Gene variants in the folate metabolic pathway (e.g., MTHFR rs1801133 (677 C > T) and MTHFD1 rs2236225 (R653Q)) have been found to increase NTD risk. We hypothesized that variants in additional folate/B12 pathway genes contribute to NTD risk.
A tagSNP approach was used to screen common variation in 82 candidate genes selected from the folate/B12 pathway and NTD mouse models. We initially genotyped polymorphisms in 320 Irish triads (NTD cases and their parents), including 301 cases and 341 Irish controls to perform case–control and family based association tests. Significantly associated polymorphisms were genotyped in a secondary set of 250 families that included 229 cases and 658 controls. The combined results for 1441 SNPs were used in a joint analysis to test for case and maternal effects.
Nearly 70 SNPs in 30 genes were found to be associated with NTDs at the p < 0.01 level. The ten strongest association signals (p-value range: 0.0003–0.0023) were found in nine genes (MFTC, CDKN2A, ADA, PEMT, CUBN, GART, DNMT3A, MTHFD1 and T (Brachyury)) and included the known NTD risk factor MTHFD1 R653Q (rs2236225). The single strongest signal was observed in a new candidate, MFTC rs17803441 (OR = 1.61 [1.23-2.08], p = 0.0003 for the minor allele). Though nominally significant, these associations did not remain significant after correction for multiple hypothesis testing.
To our knowledge, with respect to sample size and scope of evaluation of candidate polymorphisms, this is the largest NTD genetic association study reported to date. The scale of the study and the stringency of correction are likely to have contributed to real associations failing to survive correction. We have produced a ranked list of variants with the strongest association signals. Variants in the highest rank of associations are likely to include true associations and should be high priority candidates for further study of NTD risk.
PMCID: PMC3458983  PMID: 22856873
Neural tube defects; Spina bifida; Folic acid; One-carbon metabolism; Candidate gene
6.  Folate-related gene variants in Irish families affected by neural tube defects 
Frontiers in Genetics  2013;4:223.
Periconceptional folic acid use can often prevent neural tube defects (NTDs). Variants of genes involved in folate metabolism in mothers and children have been associated with occurrence of NTDs. We identified Irish families with individuals affected by neural tube defects. In these families, we observed that neural tube defects and birth defects overall occurred at a higher rate in the maternal lineage compared with the paternal lineage. The goal of this study was to look for evidence for genetic effects that could explain the discrepancy in the occurrence of these birth defects in the maternal vs. paternal lineage. We genotyped blood samples from 322 individuals from NTD-affected Irish families, identified through their membership in spina bifida associations. We looked for differences in distribution in maternal vs. paternal lineages of five genetic polymorphisms: the DHFR 19 bp deletion, MTHFD1 1958G>A, MTHFR 1298A>C, MTHFR 677C>T, and SLC19A1 80A>G. In addition to looking at genotypes individually, we determined the number of genotypes associated with decreased folate metabolism in each relative (“risk genotypes”) and compared the distribution of these genotypes in maternal vs. paternal relatives. Overall, maternal relatives had a higher number of genotypes associated with lower folate metabolism than paternal relatives (p = 0.017). We expected that relatives would share the same risk genotype as the individuals with NTDs and/or their mothers. However, we observed that maternal relatives had an over-abundance of any risk genotype, rather than one specific genotype. The observed genetic effects suggest an epigenetic mechanism in which decreased folate metabolism results in epigenetic alterations related to the increased rate of NTDs and other birth defects seen in the maternal lineage. Future studies on the etiology of NTDs and other birth defects could benefit from including multigenerational extended families, in order to explore potential epigenetic mechanisms.
PMCID: PMC3818582  PMID: 24223580
neural tube defects; folate metabolism; DHFR 19bp deletion; MTHFD1 1958G>A; MTHFR 1298A>C; MTHFR 677C>T; SLC19A1 80A>G; maternal inheritance
7.  Genotyping of a tri-allelic polymorphism by a novel melting curve assay in MTHFD1L: an association study of nonsyndromic Cleft in Ireland 
BMC Medical Genetics  2012;13:29.
Polymorphisms within the MTHFD1L gene were previously associated with risk of neural tube defects in Ireland. We sought to test the most significant MTHFD1L polymorphisms for an association with risk of cleft in an Irish cohort. This required the development of a new melting curve assay to genotype the technically challenging MTHFD1L triallelic deletion/insertion polymorphism (rs3832406).
Melting curve analysis was used to genotype the MTHFD1L triallelic deletion/insertion polymorphism (rs3832406) and a Single Nucleotide Polymorphism rs17080476 in an Irish cohort consisting of 981 Irish case-parent trios and 1,008 controls. Tests for association with nonsyndromic cleft lip with or without cleft palate and cleft palate included case/control analysis, mother/control analysis and Transmission Disequilibrium Tests of case-parent trios.
A successful melting curve genotyping assay was developed for the deletion/insertion polymorphism (rs3832406). The TDT analysis initially showed that the rs3832406 polymorphism was associated with isolated cleft lip with or without cleft palate. However, corrected p-values indicated that this association was not significant.
Melting Curve Analysis can be employed to successfully genotype challenging polymorphisms such as the MTHFD1L triallelic deletion/insertion polymorphism (DIP) reported here (rs3832406) and is a viable alternative to capillary electrophoresis. Corrected p-values indicate no association between MTHFD1L and risk of cleft in an Irish cohort.
PMCID: PMC3419639  PMID: 22520921
8.  Association of Methylenetetrahydrofolate Dehydrogenase 1 Polymorphisms with Cancer: A Meta-Analysis 
PLoS ONE  2013;8(7):e69366.
Studies investigating the association between single-nucleotide polymorphisms (SNPs) of the methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) and cancer risk report conflicting results. To derive a more precise estimation of the relationship between MTHFD1 polymorphisms and cancer risk, the present meta-analysis was carried out.
Methodology/Principal Findings
A comprehensive search was conducted to determine all the eligible studies about MTHFD1 polymorphisms and cancer risk. Combined odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the association between the MTHFD1 polymorphisms and cancer risk. We investigated by meta-analysis the effects of 2 polymorphisms in MTHFD1: G1958A (17 studies, 12348 cases, 44132 controls) and G401A (20 studies, 8446 cases, 14020 controls). The overall results indicated no major influence of these 2 polymorphisms on cancer risk. For G1958A, a decreased cancer risk was found in acute lymphoblastic leukemia (ALL)/Asians (the dominant: OR = 0.74, 95% CI = 0.58–0.94, P = 0.01; allelic: OR = 0.80, 95% CI = 0.65–0.99, P = 0.04) and other cancers (recessive: OR = 0.80, 95% CI = 0.66–0.96, P = 0.02). For G401A, the data showed that MTHFD1 G401A polymorphism was associated with a decreased colon cancer risk under dominant model (OR = 0.89, 95% CI = 0.80–0.99, P = 0.04).
The results suggest that MTHFD1 G1958A polymorphism might be associated with a decreased risk of ALL and other cancers. Meanwhile, the MTHFD1 G401A might play a protective role in the development of colon cancer. Large-scale and well-designed case-control studies are necessary to validate the risk identified in the present meta-analysis.
PMCID: PMC3716643  PMID: 23894459
9.  Roles of Genetic Polymorphisms in the Folate Pathway in Childhood Acute Lymphoblastic Leukemia Evaluated by Bayesian Relevance and Effect Size Analysis 
PLoS ONE  2013;8(8):e69843.
In this study we investigated whether polymorphisms in the folate pathway influenced the risk of childhood acute lymphoblastic leukemia (ALL) or the survival rate of the patients. For this we selected and genotyped 67 SNPs in 15 genes in the folate pathway in 543 children with ALL and 529 controls. The results were evaluated by gender adjusted logistic regression and by the Bayesian network based Bayesian multilevel analysis of relevance (BN-BMLA) methods. Bayesian structure based odds ratios for the relevant variables and interactions were also calculated. Altogether 9 SNPs in 8 genes were associated with altered susceptibility to ALL. After correction for multiple testing, two associations remained significant. The genotype distribution of the MTHFD1 rs1076991 differed significantly between the ALL and control population. Analyzing the subtypes of the disease the GG genotype increased only the risk of B-cell ALL (p = 3.52×10−4; OR = 2.00). The GG genotype of the rs3776455 SNP in the MTRR gene was associated with a significantly reduced risk to ALL (p = 1.21×10−3; OR = 0.55), which resulted mainly from the reduced risk to B-cell and hyperdiploid-ALL. The TC genotype of the rs9909104 SNP in the SHMT1 gene was associated with a lower survival rate comparing it to the TT genotype (80.2% vs. 88.8%; p = 0.01). The BN-BMLA confirmed the main findings of the frequentist-based analysis and showed structural interactional maps and the probabilities of the different structural association types of the relevant SNPs especially in the hyperdiploid-ALL, involving additional SNPs in genes like TYMS, DHFR and GGH. We also investigated the statistical interactions and redundancies using structural model properties. These results gave further evidence that polymorphisms in the folate pathway could influence the ALL risk and the effectiveness of the therapy. It was also shown that in gene association studies the BN-BMLA could be a useful supplementary to the traditional frequentist-based statistical method.
PMCID: PMC3734218  PMID: 23940529
10.  Methylenetetrahydrofolate dehydrogenase (MTHFD) enzyme polymorphism as a maternal risk factor for trisomy 21: a clinical study  
Journal of Medicine and Life  2010;3(4):454-457.
Recent reports linking Down syndrome (DS) to maternal polymorphism at the methylenetetrahydrofolate dehydrogenase (MTHFD) locus have generated a great interest among investigators in the field. In the current study, we examine one genetic polymorphism involved in homocysteine/folate pathway as a risk factor for DS in a Romanian urban–area women cohort. Our results show that the frequencies of MTHFD1 alleles, as well as the frequencies of MTHFD11958 genotypes (GG, GA, AA, GA+AA) do not correlate with DS pregnancies, demonstrating no difference between the case and control groups, as opposed to the findings of Scala et al. (2006) on an Italian cohort.
PMCID: PMC3019079  PMID: 21254748
Down syndrome; folate; methylenetetrahydrofolate dehydrogenase ; (MTHFD); MTHFD1 1958G>A
11.  Neural Tube Defects and Folate Pathway Genes: Family-Based Association Tests of Gene–Gene and Gene–Environment Interactions 
Environmental Health Perspectives  2006;114(10):1547-1552.
Folate metabolism pathway genes have been examined for association with neural tube defects (NTDs) because folic acid supplementation reduces the risk of this debilitating birth defect. Most studies addressed these genes individually, often with different populations providing conflicting results.
Our study evaluates several folate pathway genes for association with human NTDs, incorporating an environmental cofactor: maternal folate supplementation.
In 304 Caucasian American NTD families with myelomeningocele or anencephaly, we examined 28 polymorphisms in 11 genes: folate receptor 1, folate receptor 2, solute carrier family 19 member 1, transcobalamin II, methylenetetrahydrofolate dehydrogenase 1, serine hydroxymethyl-transferase 1, 5,10-methylenetetrahydrofolate reductase (MTHFR), 5-methyltetrahydrofolate-homo-cysteine methyltransferase, 5-methyltetrahydrofolate-homocysteine methyltransferase reductase, betaine-homocysteine methyltransferase (BHMT), and cystathionine-beta-synthase.
Only single nucleotide polymorphisms (SNPs) in BHMT were significantly associated in the overall data set; this significance was strongest when mothers took folate-containing nutritional supplements before conception. The BHMT SNP rs3733890 was more significant when the data were stratified by preferential transmission of the MTHFR rs1801133 thermolabile T allele from parent to offspring. Other SNPs in folate pathway genes were marginally significant in some analyses when stratified by maternal supplementation, MTHFR, or BHMT allele transmission.
BHMT rs3733890 is significantly associated in our data set, whereas MTHFR rs1801133 is not a major risk factor. Further investigation of folate and methionine cycle genes will require extensive SNP genotyping and/or resequencing to identify novel variants, inclusion of environmental factors, and investigation of gene–gene interactions in large data sets.
PMCID: PMC1626421  PMID: 17035141
folate; folic acid supplementation; genetic association; neural tube defects
12.  Fermentation of Fructose and Synthesis of Acetate from Carbon Dioxide by Clostridium formicoaceticum1 
Journal of Bacteriology  1972;109(2):626-632.
Clostridium formicoaceticum ferments fructose labeled with 14C in carbon 1, 4, 5, or 6 via the Embden Meyerhof pathway. In fermentations of fructose in the presence of 14CO2, acetate is formed labeled equally in both carbons. Extracts convert the methyl groups of 5-methyltetrahydrofolate and methyl-B12 to the methyl group of acetate in the presence of pyruvate. Formate dehydrogenase, 10-formyltetrahydrofolate synthetase, 5,10-methenyltetrahydrofolate cyclohydrolase, 5,10-methylenetetrahydrofolate dehydrogenase, and 5,10-methylenetetrahydrofolate reductase are present in extracts of C. formicoaceticum. These enzymes are needed for the conversion of CO2 to 5-methyltetrahydrofolate. It is proposed that acetate is totally synthesized from CO2 via the reactions catalyzed by the enzymes listed above and that 5-methyltetra-hydrofolate and a methylcorrinoid are intermediates in this synthesis.
PMCID: PMC285186  PMID: 5058446
13.  Mitochondrial NAD-Dependent Methylenetetrahydrofolate Dehydrogenase-Methenyltetrahydrofolate Cyclohydrolase Is Essential for Embryonic Development 
Molecular and Cellular Biology  2002;22(12):4158-4166.
Folate-dependent enzymes are compartmentalized between the cytoplasm and mitochondria of eukaryotes. The role of mitochondrial folate-dependent metabolism and the extent of its contribution to cytoplasmic processes are areas of active investigation. NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) catalyzes the interconversion of 5,10-methylenetetrahydrofolate and 10-formyltetrahydrofolate in mitochondria of mammalian cells, but its metabolic role is not yet clear. Its expression in embryonic tissues but not in most adult tissues as well as its stringent transcriptional regulation led us to postulate that it may play a role in embryonic development. To investigate the metabolic role of NMDMC, we used a knockout approach to delete the nmdmc gene in mice. Heterozygous mice appear healthy, but homozygous NMDMC knockout mice die in utero. At embryonic day 12.5 (E12.5), homozygous null embryos exhibit no obvious developmental defects but are smaller and pale and die soon thereafter. Mutant fetal livers contain fewer nucleated cells and lack the characteristic redness of wild-type or heterozygous livers. The frequencies of CFU-erythroid (CFU-E) and burst-forming unit-erythroid (BFU-E) from fetal livers of E12.5 null mutants were not reduced compared with those of wild-type or heterozygous embryos. It has been assumed that initiation of protein synthesis in mitochondria requires a formylated methionyl-tRNAfmet. One role postulated for NMDMC is to provide 10-formyltetrahydrofolate as a formyl group donor for the synthesis of this formylmethionyl-tRNAfmet. To determine if the loss of NMDMC impairs protein synthesis and thus could be a cause of embryonic lethality, mitochondrial translation products were examined in cells in culture. Mitochondrial protein synthesis was unaffected in NMDMC-null mutant cell lines compared with the wild type. These results show that NMDMC is not required to support initiation of protein synthesis in mitochondria in isolated cells but instead demonstrate an essential role for mitochondrial folate metabolism during embryonic development.
PMCID: PMC133862  PMID: 12024029
14.  Genetic variants in phosphatidylethanolamine N-methyltransferase (PEMT) and methylenetetrahydrofolate dehydrogenase (MTHFD1) influence biomarkers of choline metabolism when folate intake is restricted 
Choline is a required nutrient with roles in liver and brain function, lipid metabolism, and fetal development. Recent data suggest that choline requirements may be altered by polymorphisms in the phosphatidylethanolamine N-methyltransferase (PEMT) gene (i.e., 5465G→A; rs7946 and -744G→C; rs12325817) and in the methylenetetrahydrofolate dehydrogenase (MTHFD1) gene (i.e., 1958G→A; rs2236225). This controlled feeding study, conducted in 2000–2001, examined the effects of the PEMT and MTHFD1 genetic variants on biomarkers of choline metabolism in pre-menopausal Mexican American women (n=43) after a 7-wk period of folate restriction (135μg as dietary folate equivalents, DFE) and after a 7-wk period of folate treatment (400 and 800μg DFE/d combined). Throughout the 14-wk study choline intake remained constant at 349mg/d. The genotype frequencies of the women were 3GG, 19GA, and 21AA for PEMT G5465A; 9GG, 17GC and 17CC for PEMT G-744C; and 9GG, 21GA and 13AA for MTHFD1 G1958A. During folate restriction, homocysteine was adversely influenced by PEMT 5465AA (P=0.001 relative to the G allele) and by MTHFD1 1958AA (P=0.085 relative to 1958GG); whereas the decline in phosphatidylcholine was attenuated by PEMT -744CC (P=0.017 relative to -744GG). During folate treatment, no effects of the genotypes on the response of the measured variables were detected. These data suggest that polymorphisms in genes relevant to choline metabolism modulate parameters of choline status when folate intake is restricted. Additional studies with larger samples sizes are needed to examine the relationship between these genetic variants and varied choline intake in populations with increased demands for choline (i.e., pregnant women).
PMCID: PMC2655101  PMID: 19167960
PEMT; MTHFD1; choline; folate; genetic variants
Mammalian mitochondrial C1-tetrahydrofolate (THF) synthase (MTHFD1L gene product) is a monofunctional 10-formyl-THF synthetase, lacking the 5,10-methylene-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase activities typically found in the trifunctional cytoplasmic proteins. Here we report the submitochondrial localization of epitope-tagged human mitochondrial C1-THF synthase expressed in Chinese hamster ovary cells. Mitochondrial fractionation experiments show that human mitochondrial C1-THF synthase behaves as a peripheral membrane protein, tightly associated with the matrix side of the mitochondrial inner membrane. Inner mitochondrial membrane association was also observed for the endogenous mitochondrial C1-THF synthase in adult rat spleen. We also purified and characterized the recombinant protein product (short isoform) of the alternatively spliced short transcript of the mitochondrial isozyme. Methylene-THF dehydrogenase assays confirmed that the short isoform is not enzymatically active. The purified short isoform was used in the production of polyclonal antibodies specific for the mitochondrial isozyme. These antibodies detected endogenous full-length mitochondrial C1-THF synthase in mitochondria from adult rat spleen and human placenta, confirming the expression of the mitochondrial isozyme in adult mammalian tissues.
PMCID: PMC2707772  PMID: 18996079
16.  Inhibition of 5,10-Methenyltetrahydrofolate Synthetase 
The interaction of 5-formyltetrahydrofolate analogs with murine methenyltetrahydrofolate synthetase (MTHFS) was investigated using steady-state kinetics, molecular modeling, and site-directed mutagenesis. MTHFS catalyzes the irreversible cyclization of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Folate analogs that cannot undergo the rate-limiting step in catalysis were inhibitors of murine MTHFS. 5-formyltetrahydrohomofolate was an effective inhibitor of murine MTHFS (Ki = 0.7 μM), whereas 5-formyl, 10-methyltetrahydrofolate was a weak inhibitor (Ki = 10 μM). The former, but not the latter, was slowly phosphorylated by MTHFS. 5-formyltetrahydrohomofolate was not a substrate for murine MTHFS, but was metabolized when the MTHFS active site Y151 was mutated to Ala. MTHFS active site residues do not directly facilitate N10 attack on the on the N5-iminium phosphate intermediate, but rather restrict N10 motion around N5. Inhibitors specifically designed to block N10 attack appear to be less effective than the natural 10-formyltetrahydrofolate polyglutamate inhibitors.
PMCID: PMC1850231  PMID: 17250800
17.  Neural tube defects induced by folate deficiency in mutant curly tail (Grhl3) embryos are associated with alteration in folate one-carbon metabolism but are unlikely to result from diminished methylation 
Folate one-carbon metabolism has been implicated as a determinant of susceptibility to neural tube defects (NTDs), owing to the preventive effect of maternal folic acid supplementation and the higher risk associated with markers of diminished folate status.
Folate one-carbon metabolism was compared in curly tail (ct/ct) and genetically matched congenic (+ct/+ct) mouse strains using the deoxyuridine suppression test in embryonic fibroblast cells and by quantifying s-adenosylmethionine (SAM) and s-adenosylhomocysteine (SAH) in embryos using liquid chromatography tandem mass spectrometry. A possible genetic interaction between curly tail and a null allele of 5,10-methylenetetrahydrofolate reductase (MTHFR) was investigated by generation of compound mutant embryos.
There was no deficit in thymidylate biosynthesis in ct/ct cells but incorporation of exogenous thymidine was lower than in +ct/+ct cells. In +ct/+ct embryos the SAM/SAH ratio was diminished by dietary folate deficiency and normalised by folic acid or myor-inositol treatment, in association with prevention of NTDs. In contrast, folate deficiency caused a significant increase in SAM/SAH ratio in ct/ct embryos. Loss of MTHFR function in curly tail embryos significantly reduced the SAM/SAH ratio but did not cause cranial NTDs or alter the frequency of caudal NTDs.
Curly tail fibroblasts and embryos, in which Grhl3 expression is reduced, display alterations in one-carbon metabolism, particularly in the response to folate deficiency, compared with genetically-matched congenic controls in which Grhl3 is unaffected. However, unlike folate deficiency, diminished methylation potential appears to be insufficient to cause cranial NTDs in the curly tail strain, and neither does it increase the frequency of caudal NTDs.
PMCID: PMC3634156  PMID: 20589880
Neural tube defects; folate; methylation; curly tail; inositol
18.  Genetic variants in the folate pathway and risk of childhood acute lymphoblastic leukemia 
Cancer causes & control : CCC  2011;22(9):1243-1258.
Folate is involved in the one-carbon metabolism that plays an essential role in the synthesis, repair and methylation of DNA. We examined whether child’s germline genetic variation in the folate pathway is associated with childhood acute lymphoblastic leukemia (ALL), and whether periconception maternal folate and alcohol intake modify the risk.
Seventy-six single nucleotide polymorphisms (SNPs), including 66 haplotype-tagging SNPs in 10 genes (CBS, DHFR, FOLH1, MTHFD1, MTHFR, MTR, MTRR, SHMT1, SLC19A1, and TYMS) were genotyped in 377 ALL cases and 448 controls. Log-additive associations between genotypes and ALL risk were adjusted for age, sex, Hispanic ethnicity (when appropriate), and maternal race.
Single and haplotype SNPs analyses showed statistically significant associations between SNPs located in (or adjacent to) CBS, MTRR, TYMS/ENOFS and childhood ALL. Many regions of CBS were associated with childhood ALL in Hispanics and non-Hispanics (P <0.01). Levels of maternal folate intake modified associations with SNPs in CBS, MTRR, and TYMS.
Our data suggest the importance of genetic variability in the folate pathway and childhood ALL risk.
PMCID: PMC3744066  PMID: 21748308
Case-control study; Children; DNA methylation; Folate; Genetic polymorphisms; Leukemia
19.  Genetic polymorphisms in one-carbon metabolism: associations with CpG island methylator phenotype (CIMP) in colon cancer and the modifying effects of diet 
Carcinogenesis  2007;28(8):1672-1679.
This study investigated associations between CpG island methylator phenotype (CIMP) colon cancer and genetic polymorphisms relevant to one-carbon metabolism and thus, potentially the provision of methyl groups and risk of colon cancer. Data from a large, population-based case–control study (916 incident colon cancer cases and 1972 matched controls) were used. Candidate polymorphisms in methylenetetrahydrofolate reductase (MTHFR), thymidylate synthase (TS), transcobalamin II (TCNII), methionine synthase (MTR), reduced folate carrier (RFC), methylene-tetrahydrofolate dehydrogenase 1 (MTHFD1), dihydrofolate reductase (DHFR) and alcohol dehydrogenase 3 (ADH3) were evaluated. CIMP− or CIMP+ phenotype was based on five CpG island markers: MINT1, MINT2, MINT31, p16 and MLH1. The influence of specific dietary factors (folate, methionine, vitamin B12 and alcohol) on these associations was also analyzed. We hypothesized that polymorphisms involved in the provision of methyl groups would be associated with CIMP+ tumors (two or more of five markers methylated), potentially modified by diet. Few associations specific to CIMP+ tumors were observed overall, which does not support the hypothesis that the provision of methyl groups is important in defining a methylator phenotype. However, our data suggest that genetic polymorphisms in MTHFR 1298A > C, interacting with diet, may be involved in the development of highly CpG-methylated colon cancers. AC and CC genotypes in conjunction with a high-risk dietary pattern (low folate and methionine intake and high alcohol use) were associated with CIMP+ (OR = 2.1, 95% CI = 1.3–3.4 versus AA/high risk; P-interaction = 0.03). These results provide only limited support for a role of polymorphisms in one-carbon metabolism in the etiology of CIMP colon cancer.
PMCID: PMC2442467  PMID: 17449906
20.  Purification and characterization of NADP(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Peptostreptococcus productus marburg. 
Journal of Bacteriology  1991;173(4):1414-1419.
The 5,10-methylenetetrahydrofolate dehydrogenase of heterotrophically grown Peptostreptococcus productus Marburg was purified to apparent homogeneity. The purified enzyme catalyzed the reversible oxidation of methylenetetrahydrofolate with NADP+ as the electron acceptor at a specific activity of 627 U/mg of protein. The Km values for methylenetetrahydrofolate and for NADP+ were 27 and 113 microM, respectively. The enzyme, which lacked 5,10-methenyltetrahydrofolate cyclohydrolase activity, was insensitive to oxygen and was thermolabile at temperatures above 40 degrees C. The apparent molecular mass of the enzyme was estimated by gel filtration to be 66 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a single subunit of 34 kDa, accounting for a dimeric alpha 2 structure of the enzyme. Kinetic studies on the initial reaction velocities with different concentrations of both substrates in the absence and presence of NADPH as the reaction product were interpreted to indicate that the enzyme followed a sequential reaction mechanism. After gentle ultracentrifugation of crude extracts, the enzyme was recovered to greater than 95% in the soluble (supernatant) fraction. Sodium (10 microM to 10 mM) had no effect on enzymatic activity. The data were taken to indicate that the enzyme was similar to the methylenetetrahydrofolate dehydrogenases of other homoacetogenic bacteria and that the enzyme is not involved in energy conservation of P. productus.
PMCID: PMC207278  PMID: 1899860
21.  The crystal structure of Leishmania major N5,N10-methylenetetrahydrofolate dehydrogenase/cyclohydrolase and assessment of a potential drug target☆ 
Molecular and Biochemical Parasitology  2012;181(2-6):178-185.
Graphical abstract
The crystal structure of Leishmania major N5,N10-methylenetetrahydrofolate dehydrogenase/N5,N10-methenyltetrahydrofolate cyclohydrolase is used to assess the potential of this bifunctional enzyme as a drug target.
► We report the structure of Leishmania major methylenetetrahydrofolate dehydrogenase/cyclohydrolase. ► Sequence–structure comparisons are carried out with homologues from kinetoplastids and the human host. ► The potential of this bifunctional enzyme as a drug target is assessed. ► The similarities between parasite and human enzymes suggest a difficult target for drug discovery.
Three enzyme activities in the protozoan Leishmania major, namely N5,N10-methylenetetrahydrofolate dehydrogenase/N5,N10-methenyltetrahydrofolate cyclohydrolase (DHCH) and N10-formyltetrahydrofolate ligase (FTL) produce the essential intermediate N10-formyltetrahydrofolate. Although trypanosomatids possess at least one functional DHCH, the same is not true for FTL, which is absent in Trypanosoma brucei. Here, we present the 2.7 Å resolution crystal structure of the bifunctional apo-DHCH from L. major, which is a potential drug target. Sequence alignments show that the cytosolic enzymes found in trypanosomatids share a high level of identity of approximately 60%. Additionally, residues that interact and participate in catalysis in the human homologue are conserved amongst trypanosomatid sequences and this may complicate attempts to derive potent, parasite specific DHCH inhibitors.
PMCID: PMC3368264  PMID: 22108435
Antifolate; Cyclohydrolase; Dehydrogenase; Drug target; Leishmania; Trypanosoma
22.  Methylenetetrahydrofolate reductase and transcobalamin genetic polymorphisms in human spontaneous abortion: biological and clinical implications 
The pathogenesis of human spontaneous abortion involves a complex interaction of several genetic and environmental factors. The firm association between increased homocysteine concentration and neural tube defects (NTD) has led to the hypothesis that high concentrations of homocysteine might be embryotoxic and lead to decreased fetal viability. There are several genetic polymorphisms that are associated with defects in folate- and vitamin B12-dependent homocysteine metabolism. The methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C polymorphisms cause elevated homocysteine concentration and are associated with an increased risk of NTD. Additionally, low concentration of vitamin B12 (cobalamin) or transcobalamin that delivers vitamin B12 to the cells of the body leads to hyperhomocysteinemia and is associated with NTD. This effect involves the transcobalamin (TC) 776C>G polymorphism. Importantly, the biochemical consequences of these polymorphisms can be modified by folate and vitamin B12 supplementation. In this review, I focus on recent studies on the role of hyperhomocysteinemia-associated polymorphisms in the pathogenesis of human spontaneous abortion and discuss the possibility that periconceptional supplementation with folate and vitamin B12 might lower the incidence of miscarriage in women planning a pregnancy.
PMCID: PMC356929  PMID: 14969589
23.  Mthfs is an Essential Gene in Mice and a Component of the Purinosome 
Tetrahydrofolates (THF) are a family of cofactors that function as one-carbon donors in folate-dependent one-carbon metabolism, a metabolic network required for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine in the cytoplasm. 5-FormylTHF is not a cofactor in one-carbon metabolism, but serves as a storage form of THF cofactors. 5-formylTHF is mobilized back into the THF cofactor pool by methenyltetrahydrofolate synthetase (MTHFS), which catalyzes the irreversible and ATP-dependent conversion 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Mthfs is not an essential gene in Arabidopsis, but MTHFS expression is elevated in animal tumors, enhances de novo purine synthesis, confers partial resistance to antifolate purine synthesis inhibitors and increases rates of folate catabolism in mammalian cell cultures. However, the mechanisms underlying these effects of MTHFS expression have yet to be established. The purpose of this study was to investigate the role and essentiality of MTHFS in mice. Mthfs was disrupted through the insertion of a gene trap vector between exons 1 and 2. Mthfsgt/+ mice were fertile and viable. No Mthfsgt/gt embryos were recovered from Mthfsgt/+ intercrosses, indicating Mthfs is an essential gene in mice. Tissue MTHFS protein levels are decreased in both Mthfsgt/+ and Mthfs+/+ mice placed on a folate and choline deficient diet, and mouse embryonic fibroblasts from Mthfsgt/+ embryos exhibit decreased capacity for de novo purine synthesis without impairment in de novo thymidylate synthesis. MTHFS was shown to co-localize with two enzymes of the de novo purine synthesis pathway in HeLa cells in a cell cycle-dependent manner, and to be modified by the small ubiquitin-like modifier (SUMO) protein. Mutation of the consensus SUMO modification sites on MTHFS eliminated co-localization of MTHFS with the de novo purine biosynthesis pathway under purine-deficient conditions. The results from this study indicate that MTHFS enhances purine biosynthesis by delivering 10-formylTHF to the purinosome in a SUMO-dependent fashion.
PMCID: PMC3268590  PMID: 22303332
folate; MTHFS; purinosome; SHMT; 5-formyltetrahydrofolate; purine biosynthesis; leucovorin; SUMO
24.  Evaluation of 64 Candidate Single Nucleotide Polymorphisms as Risk Factors for Neural Tube Defects in a Large Irish Study Population 
Individual studies of the genetics of neural tube defects (NTDs) contain results on a small number of genes in each report. To identify genetic risk factors for NTDs, we evaluated potentially functional single nucleotide polymorphisms (SNPs) that are biologically plausible risk factors for NTDs but that have never been investigated for an association with NTDs, examined SNPs that previously showed no association with NTDs in published studies, and tried to confirm previously reported associations in folate-related and non-folate-related genes. We investigated 64 SNPs in 34 genes for association with spina bifida in up to 558 case-families (520 cases, 507 mothers, 457 fathers) and 994 controls in Ireland. Case-control and mother-control comparisons of genotype frequencies, tests of transmission disequilibrium, and log-linear regression models were used to calculate effect estimates. Spina bifida was associated with over-transmission of the LEPR (leptin receptor) rs1805134 minor C allele (genotype relative risk (GRR): 1.5; 95% confidence interval (CI): 1.0, 2.1; P = 0.0264) and the COMT (catechol-O-methyltransferase) rs737865 major T allele (GRR: 1.4; 95% CI: 1.1, 2.0; P = 0.0206). After correcting for multiple comparisons, these individual test P-values exceeded 0.05. Consistent with previous reports, spina bifida was associated with MTHFR 677C>T, T (Brachyury) rs3127334, LEPR K109R, and PDGFRA promoter haplotype combinations. The associations between LEPR SNPs and spina bifida suggest a possible mechanism for the finding that obesity is a NTD risk factor. The association between a variant in COMT and spina bifida implicates methylation and epigenetics in NTDs.
PMCID: PMC3503244  PMID: 21204206
congenital abnormalities; folic acid; neural tube defects; single nucleotide polymorphism; spina bifida
25.  Gene Variants in the Folate-mediated One-carbon Metabolism (FOCM) Pathway as Risk Factors for Conotruncal Heart Defects 
We evaluated 35 variants among four folate-mediated one-carbon metabolism pathway genes, MTHFD1, SHMT1, MTHFR, and DHFR as risk factors for conotruncal heart defects. Cases with a diagnosis of single gene disorders or chromosomal aneusomies were excluded. Controls were randomly selected from area hospitals in proportion to their contribution to the total population of live-born infants. Odds Ratios (OR) and the 95% confidence intervals were computed for each genotype (homozygous variant or heterozygote, versus homozygous wildtype) and for increase of each less common allele (log-additive model). Interactions between each variant and three folate intake variables (maternal multivitamin use, maternal dietary folate intake, and combined maternal folate intake) were also evaluated under the log-additive model. In general, we did not identify notable associations. The A allele of MTHFD1 rs11627387 was associated with a 1.7-fold increase in conotruncal defects risk in both Hispanic mothers (OR=1.7, 95% CI=1.1∼2.5) and Hispanic infants (OR=1.7, 95% CI=1.2∼2.3). The T allele of MTHFR rs1801133 was associated with a 2.8-fold increase of risk among Hispanic women whose dietary folate intake was ≤ 25th centile. The C allele of MTHFR rs1801131 was associated with a two-fold increase of risk (OR=2.0, 95%CI=1.0∼3.9) only among those whose dietary folate intake was >25th centile. Our study suggested that MTHFD1 rs11627387 may be associated with risk of conotruncal defects through both maternal and offspring genotype effect among the Hispanics. Maternal functional variants in MTHFR gene may interact with dietary folate intake and modify the conotruncal defects risk in the offspring.
PMCID: PMC3331895  PMID: 22495907
conotruncal defects; one carbon metabolism; folate; MTHFR; MTHFD1; DHFR; SHMT1

Results 1-25 (395950)